PROJECT SUMMARY/ABSTRACT Herpes Simplex viruses (HSV-1 and HSV-2) enter different cells by at least three different routes, which include direct fusion at the plasma membrane, pH-dependent endocytosis, and pH-independent endocytosis. Understanding HSV entry routes has important implications for its pathogenesis because HSV enters its cellular targets, epithelial cells and neurons, by different routes. HSV has at least 15 envelope proteins. Entry by any route requires four glycoproteins ? gB, gD, gH, and gL, termed essential ? and a host gD receptor. But, what guides the selection of specific entry routes is unclear. Single genetic deletions of HSV envelope proteins outside of the essential set of four have modest entry phenotypes, and as the result, these 11 proteins are typically referred to as ?non-essential? with regard to entry. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by the presence of functional redundancies among the many HSV-1 envelope proteins or their cell-specific contributions. Therefore, accurate assessment of contributions of the so-called non-essential envelope proteins to HSV-1 entry requires an approach that enables systematic gain-of-function experiments. We generated Vesicular Stomatitis Virus (VSV) virions pseudotyped with essential HSV-1 entry glycoproteins gB, gD, gH, and gL and lacking the native VSV fusogen G (VSV?G-BHLD). Like HSV-1, VSV?G-BHLD pseudotypes enter gD-receptor-bearing cells. But, unlike HSV- 1, they enter these cell types by pH-dependent endocytosis, rather than by fusion at the plasma membrane or pH-independent endocytosis, and with variable efficiency. We hypothesize that the core HSV-1 entry machinery ? gB, gD, gH, and gL ? facilitates entry into all gD-receptor-bearing cell types by pH-dependent endocytosis with variable efficiency while the so-called non-essential HSV-1 envelope proteins enable entry by other routes as well as enhance entry, in cell-type-dependent manner. This hypothesis will be examined in two Specific Aims: Aim 1. To determine contributions of the four essential HSV-1 glycoproteins to HSV-1 tropism and the selection of entry routes using VSV?G-BHLD pseudotypes. Aim 2. To determine contributions of HSV-1 envelope proteins outside the essential four to entry efficiency and the choice of entry route in a series of gain-of-function experiments utilizing the VSV?G-BHLD platform. The scientific premise of the proposed work is that VSV pseudotyping system provides an exciting new tool for dissecting contributions of HSV envelope proteins ? both the essential four and the rest ? to HSV tropism and entry routes. The proposal is significant because it addresses an important problem that has impeded the unraveling of the cell entry mechanism of HSV. The proposal is innovative due to its use of VSV pseudotyped with HSV glycoproteins and the opportunity for systematic gain-of-function studies to identify contributions of the so-called non-essential envelope proteins in HSV entry.